Carotenoids are widespread in nature a kind of yellow to red substance. In different fruits and vegetables, there are different types of carotenoids, such as β-carotene in carrots, lutein in marigold flowers, zeaxanthin in strawberry, lycopene in tomato, capsanthin and capsochrome in chili class plants. In some dark fruit and vegetables, egg, fish, crustaceans, birds, algae and bacteria more carotenoids are existed, wherein the content of lutein is higher. In recent years, a number of animal and human trials showed that the beneficial effects of carotenoids. Carotenoids may be generally divided into the two sub-categories, namely, a relatively strong polarity of xanthophylls or oxygenated carotenoids, such as lutein, zeaxanthin, astaxanthin, and the non-polar of hydrocarbon carotenoids, such as β-carotene, lycopene. These two carotenoids of subclass, each contains at least nine conjugated double bonds, which not only gives the color features of carotenoid, also has strong antioxidant function in disease control, they can stop or prevent, like cancer, arteriosclerosis, cataracts, pigmentation and other degenerative diseases. And because carotenoids efficient scavenging of reactive oxygen free radicals and prevent free radicals generated, they can limit the damaging effects of oxidative free radicals.
For all carotinoid, due to their potential capability to prevent one kind of age related macular degenerations (ARMD), scientists and the public have paid more and more attention to the xanthophyll and zeaxanthin. Xanthophyll and zeaxanthin are the only two types of carotinoid existing within the spot area (macular degeneration) of human being's retina, and the area is closely related to the visual sensitivity of human beings (Bone et al. Invest. Ophthamal. Vis. Sci. 34:2033-2040, 1993). Usually eating of fruits and vegetables that are rich in xanthophyll and zeaxanthin can reduce the risk of getting aged macular degeneration by 43% (Seddon et al. J. Am. Med. Assoc. 272:1413-1420, 1994), and the metabolic pathway of xanthophyll and zeaxanthin for preventing aged macular degeneration has been cleared at present. The Food and Drug Administration also considers xanthophyll and zeaxanthin as (GARS), it means “generally recognized as safe”. Therefore, these carotinoid can be used by themselves or together with other materials as nutritional supplements and food coloring agents, as well as can be used clinically for preventing aged macular degeneration and cancer, etc.
The constitutional formulas of xanthophyll and zeaxanthin are as follows respectively, they are isomers, the only difference between zeaxanthin and xanthophyll is the position of double bonds on one (not two) end ring, the position of the double bonds on the two end rings of the former is symmetrical, but that of the latter is asymmetrical, i.e. the straight chain part of the xanthophyll and zeaxanthin′ each molecule is the conjugation structure having double bonds and single bond alternatively. In the molecule of zeaxanthin, the molecule conjugation structure extends to the first bond on the two end rings, but the degree of conjugation structure of xanthophyll is lower, because its double bonds on one of its end rings do not form a correct arrangement as a complete conjugation structure, and the difference of molecular structure results in xanthophyll and zeaxanthin have some differences in function properties.

As a kind of natural pigments, xanthophyll and zeaxanthin exist widely in nature, they mainly exist in higher plants, algae, fishes, shell class and bacterias, and they exist in the forms of ester in the body of living beings. Among these living beings, the marigold flower is a kind of good source for xanthophyll and zeaxanthin, it has about 2 g xanthophyll class materials in 100 g marigold fresh flowers, in which xanthophyll is more than 90%, and the rest are zeaxanthin and a few other carotinoid. It is the same as marigold flowers, in other kinds of higher plant and algae sources, the proportion of xanthophyll is more than zeaxanthin, but in corns the proportion of zeaxanthin is more than xanthophyll. Further, in the molecular structure stereoisomerism is presented in the xanthophyll and the zeaxanthin, different sources have different stereoisomerisms, for example, (3R,3′R,6′R)-xanthophyll and (3R,3′R)-zeaxanthin in plant resources, but xanthophyll is existed in forms of (3R,3′R,6′R)-, (3R,3′R,6′S)-, and (3R,3′S,6′S)- in animal resources, such as in fishes and shells.
Since the chemical synthesis lutein involves multi-step reaction, time-consuming, costly. Economical and easy method for large scale production of xanthophyll crystals is extracted, isolated and purified from natural sources. Many vegetables and fruits, such as spinaches, broccolis, cabbages and corns, etc, are quite rich in xanthophyll, but marigold flowers and calendulas are the richest sources of xanthophyll, of course, there also exists other carotinoid in these plants. The xanthophyll in plants usually exists in forms of single ester and double ester which usually is formed through esterification between the xanthophyll and some C12-C18 long-chain aliphatic acids.
Generally, we use organic solvents to extract xanthophyll ester from plants, preferentially from marigold flowers, calendulas and other dark green vegetables, and these organic solvents are quite easy to be separated. The marigold peaberry extract (marigold oleoresin) is a very good xanthophyll ester source, and the other classes of carotinoid are relatively not rich in it. After hydrolyzed under alkaline conditions, the xanthophyll crystals will be dissociated, wash off the fatty acid salt gotten from the soap-dissolving process, the xanthophyll crystals is further purified.
Meanwhile, as the above description, since the conjugated double bond in the molecular structure, which makes the zeaxanthin having stronger antioxidant activity than the xanthophyll, and the zeaxanthin takes a important effect for the health of human eyes. In fact, some researches in the middle to late 1980s had also proved that they mainly were zeaxanthin in the small area at the center of human eyes' macula lutea. They leave the concave concentrically and get close to the circum of the macula lutea, so the quantity of zeaxanthin gradually becomes less, and the quantiy of xanthophyll gradually becomes more. At the circum of the macula lutea, xanthophyll is the main xantheins.
This also can be found from the proportion change between xanthophyll and zeaxanthin in different parts of natural and human tissues, in the marigold flowers, the raw materials of xanthophyll, the proportion between xanthophyll and zeaxanthin is about 10-12:1, the proportion is about 3-5:1 in human blood, the proportion is 3:1 at the circum of retina macula lutea, but at the center of macula lutea the proportion is completely opposite, and it is 1:3. The recent researches have found that in an isomer of zeaxanthin at the central area if macula lutea, the internal compensation (3R,3′S,meso)-zeaxanthin took quite a lot proportion, and the proportion will be less, if it is closer to the periphery. More and more evidences have proved that the meso-zeaxanthin in macula lutea is gotten through lutein by epimerization transposition resulting, because it almost can not detect this kind of isomers of zeaxanthin in nature, human blood and other human tissues.
The distribution proportion change of xanthophyll and zeaxanthin in human eyes just proved the important and unique effects of zeaxanthin, in particular, the internal compensation zeaxanthin, for the health of human eyes. Actually, many human experiments also have proved that it will take a better effect for the complex use of xanthophyll and zeaxanthin, so most of the eye care products that contain xanthophyll on the market usually are added with certain quantity of extra zeaxanthin, i.e. it adds with xanthophyll and zeaxanthin respectively according to certain proportion in the application partition. However, the respective addition of xanthophyll and zeaxanthin must bring many unnecessary troubles for the purchase of raw materials, the operation of production process and product quality control. If it can be realized that some part of the xanthophyll forms zeaxanthin through epimerization during the process of making xanthophyll, thereby the result product can contain xanthophyll and zeaxanthin and keep the required proportion. Thus, it can just add one kind of carotinoid agents that contains certain proportion of xanthophyll and zeaxanthin, and this will result in much convenience for the subsequent product application process.
At present, there also are some patents and essays that refer to the method to prepare xanthophyll crystals in large scale from the peaberry of marigold or the method to gain zeaxanthin crystals through the epimerization transposition of xanthophyll. The targets of these essays generally are to get quite pure crystal forms of xanthophyll or zeaxanthin, and they refer to several separation steps.
U.S. Pat. No. 5,382,714 describes to separate and purify xanthophyll through washing the soap-dissolved marigold oleoresin under a quite low temperature and crystallizing the mixed solvent under a low temperature. The purification process not only is time-consuming, but also uses chlorinated organic solvents, so the products gained are not fit for being used in foods and medicines. The proportion between xanthophyll and zeaxanthin in the products do not change much compared with the raw materials.
U.S. Pat. No. 5,648,564 describes a method to separate xanthophyll crystals, it soap-dissolves xanthophyll diester-containing the propylene glycol solution of marigold oleoresins at first, and then it will recrystallize them. There are also several defects in this process: first, since the viscosity of propylene glycol is quite strong, it requires a quite high temperature during the soap-dissolving process or the following process, the entire system needs to be kept above 70° C. for about 10 hours, this obviously is disadvantageous for the stability of xanthophyll, the cis-trans isomers of xanthophyll also will change, what's more, the following separation processes, such as centrifugation and filtration, are also quite difficult; second, the collection rate of xanthophyll is quite low, it only is about 59%, and the xanthophyll content in the products is also not high.
U.S. Pat. No. 6,262,284 describes a method to use tetrahydrofuran to extract and soap-dissolve carotinoid from marigold dry flowers, it uses plenty of organic solvents during the process, these solvents are harmful to the stability of xanthophyll, and it may result in deterioration that is caused by overoxidation.
U.S. Pat. No. 6,329,557 describes a method to extract xanthophyll crystals from marigold oleoresins through large industrial scale. The defect of the process is to use plenty of organic solvents, such as normal hexanes, ketones, etc, and these solvents are not fit for using in foods.
U.S. Pat. No. 6,380,442 reports a method to separate carotinoid from plants, the method is also not attractive for industrial production, because it will use a lot of water (at least 30 times the raw materials) during the production process, and the operation is quite difficult.
U.S. Pat. No. 6,743,953 describes to use organic solvents to separate and purify xanthophyll from marigold oleoresins. It uses several organic solvents, such as ipa, ethyl acetate, normal hexane, acetone and methanol, etc, during the process, the operation is miscellaneous, the organic solvent consumption is quite large, and the collection rate is low. Therefore, this method is also not fit for industrial production.
In U.S. Pat. No. 7,271,298, it makes public a method that uses absolute ethyl alcohol as the solvent to get higher content xanthophyll crystals in high collection rate through simple technology, and this method also do not consider to enhance the proportion of zeaxanthin for the gained crystals.
U.S. Pat. No. 5,780,693 designs a routine to produce zeaxanthin taking xanthophyll as the raw materials. In general, it mainly uses dimethyl sulfoxide or the mixture of dimethyl sulfoxide and saturated alkane and/or arene organic solvents as the solvents and uses alkali hydroxides as catalysts to produce zeaxanthin through transposition of xanthophyll. Furthermore, it uses the organic solvents, such as normal hexanes, normal heptanes, dichloromethane, methyl alcohols, etc, during the reaction process. It is obviously improper to use these toxic solvents to produce food grade or medicine grade zeaxanthin.
U.S. Pat. No. 7,485,738 describes to use xanthophyll as the raw materials to gain high purity internal compensation zeaxanthin through epimerization with the catalysis of strong organic alkali, and the gained zeaxanthin crystals through this technology do not contain or contain very few xanthophyll.
The described methods in the above patents have several defects as follows: 1) using some toxic organic solvents during the processes, it is quite difficult or impossible to remove these solvents completely, and this results in that the produced xanthophyll or zeaxanthin crystals are unfit for being used as edible products of human beings; 2) or the used organic solvents are quite high in viscosity, the operation during the process of separation and purification is quite difficult, in order to gain higher content crystals, it needs a treatment process with several steps, so it is not suitable for industrial production; 3) or the collection rate of carotinoid is quite low, since it refers to several steps during the process, it results in the low product collection rate, and it is only about 50%. In particular, when using marigold flowers as the raw materials to gain the mixtures of zeaxanthin and xanthophyll through isomerization reaction to transform xanthophyll crystals partially after it gained xanthophyll crystals through soap-dissolving reaction, separation and retification, the collection rate is lower. 4) or the single concentration of xanthophyll or zeaxanthin in the products is quite high, they can not reach the purpose to adjust xanthophyll and zeaxanthin concentration through controlling the reaction process according to the requirements.
Therefore, it is necessary to find a method that is suitable to produce high purity xanthophyll and zeaxanthin in industrial scale, which uses as few as possible toxic organic solvents, refers to as few as possible steps, owns quite high collection rate, and can control the reaction parameters according to the needs, so that it can reach the purpose to adjust the xanthophyll and zeaxanthin concentration in the products.